1. Field of the Invention
As known in the art, the epidermis and dermis of mammalian skin contain different cell types, perform different functions, and have different chemical compositions. A particularly important difference between these layers is their lipid concentrations. The dermis contains fibroblasts which produce collagen and other proteins, but very little lipid. The epidermis, on the other hand, contains keratinocytes which, among other things, produce lipids, but essentially no collagen. The collagen produced by the fibroblasts provides tensile strength to the skin. The lipids produced by the kerotinocytes provide a barrier between the living tissue and the outside world.
The present invention relates to modifying the lipid content of skin for purposes of altering appearance, improving function, improving vitality, reversing the effects of aging, reversing the effects of photodamage, or treating disease by topical administration of ursolic acid, ursolic acid analogs, derivatives of ursolic acid, derivatives of ursolic acid analogs, or combinations thereof. Because the skin lipids are located in the epidermis, this modification of the lipid content of the skin takes place in that layer. For ease of reference, ursolic acid, ursolic acid analogs, derivatives of ursolic acid, derivatives of ursolic acid analogs, or combinations thereof will be referred to herein as simply a “ursolic acid compound”. The ursolic acid compound can be encapsulated in liposomes or administered in other formulations suitable for topical administration.
2. Description of Related Art
A. Patents
U.S. Pat. No. 4,857,554, Methods for Treatment of Psoriasis, is directed to treating psoriasis by applying an ointment containing ursolic acid and oleanolic acid dispersed in a petroleum jelly/lanolin carrier.
U.S. Pat. No. 4,530,934, Pharmaceutically Active Ursolic Acid Derivatives, is directed to using active derivatives of ursolic acid to treat ulcers.
U.S. Pat. No. 3,903,089, Ursolic Acid Derivatives, is directed to the synthesis of ursolic acid derivatives and analogs.
U.S. Pat. No. 5,624,909, Derivatives of Triterpenoid Acids as Inhibitors of Cell-adhesion Molecules ELAM-1 (e-selectin) and LECAM-1 (I-selectin), is directed to alleviating inflammation by administration of triterpenoid acid derivatives.
U.S. Pat. No. 5,314,877, Water-soluble Pentacyclic Triterpene Composition and Method for Producing the Same, is directed to making ursolic acid, oleanolic acid, and related triterpenoids soluble in water by formulation in cyclodextrins.
U.S. Reissue Patent RE036068, Methods for Treatment of Sundamaged Human Skin with Retinoids, is directed to reversing the effects of photodamage by topical application of retinoids.
U.S. Pat. No. 5,051,449, Treatment of Cellulite with Retinoids, is directed towards retarding or reversing cellulite accumulation in skin by topical application of retinoids.
U.S. Pat. No. 5,556,844, Pharmaceutical or Cosmetic Composition Containing a Combination of a Retinoid and a Sterol, is directed towards treatment of disorders of epidermial keratinization, epithelial proliferation, or disorders of sebaceous function by topical application of retinoids.
U.S. Pat. No. 5,075,340, Retinoic Acid Glucuronide Preparations for Application to the Skin, is directed towards treatment of acne or wrinkled skin and prevention of retinoid dermatitis by topical application of retinoic acid glucoronides.
U.S. Pat. No. 5,837,224, Method of Inhibiting Photoaging of Skin, is directed to reversing the effects of photodamage by topical application of agents that inhibit UVB-inducible matrix metalloproteinase.
B. Publications
Tokuda, H., H. Ohigashi, K. Koshimizu, and Y. Ito. 1986. Inhibitory effects of ursolic and oleanolic acid on skin tumor promotion by 12-O-tetradecanoyhlphorbol-13-acetate. Cancer Lett. 33:279–285.
Ponec, M., and A. Weerheim. 1990. Retinoids and lipid changes in keratinocytes. Meth. Enzymol. 190:30–41.
Griffiths, C. E. M., A. N. Russman, G. Majmudar, R. S. Singer, T. A. Hamilton, and J. J. Voorhees. 1993. Restoration of collagen formation in photodamaged human skin by tretinoin (retinoic acid). New Engl. J. Med. 329:530–5.
Kligman, A. M., and J. J. Leyden. 1993. Treatment of photoaged skin with topical tretinoin. Skin Pharmacol. 6 (Suppl.1):78–82).
Huang, M.-T., C.-T. Ho, Z. Y. Wang, T. Ferraro, Y.-R. Lou, K. Stauber, W. Ma, C. Georgiadis, J. D. Laskin, and A. H. Conney. 1994. Inhibition of skin tumorigenesis by rosemary and its constituents carnosol and ursolic acid. Cancer Res. 54:701–708.
Liu, J. 1995. Pharmacology of oleanolic acid and ursolic acid. J. Ethanopharmacol. 49:57–68.
Manez, S., C., C. Recio, R. M. Giner, and J.-L. Rios. 1997. Effect of selected triterpenoids on chronic dermal inflammation. Eur. J. Pharmacol. 334:103–105.
Ponec, M., A. Weerheim, J. Kempenaar, A. Mulder, G. S. Gooris, J. Bouwstra, and A. M. Mommaas. 1997. The formation of competent barrier lipids in reconstructed human epidermis requires the presence of Vitamin C. J. Invest. Dermatol. 109:348–355.
Griffiths, C. E. M. 1999. Drug treatment of photoaged skin. Drugs & Aging 14:289–301.
Japanese Patent Publication No. 11–5727, published Jan. 12, 1999, describes the use of ursolic acid in combination with retinols in a final cosmetic product to increase dermal collagen. As discussed above, collagen is located and produced in the dermis by fibroblasts. The present invention, on the other hand, is concerned with modifying the content of lipids located and produced in the epidermis by kerotinocytes.
3. Epidermal Livid Composition and Alterations During Differentiation
The epidermis of skin contains a number of lipids that are altered during differentiation as follows (see Downing et al., 1993, p210–221, In: Dermatology in General Medicine):                (i) Phospholipids: Most common are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and sphingomyelin. Phospholipids are the most abundant lipids in the basal layers of the epidermis, but decrease towards the surface of the skin so much so that they are one of the least abundant lipids in the cornified layer. Thus, the phospholipid content of keratinocytes decreases as they differentiate. Conversely, high phospholipid levels are associated with keratinocyte proliferation.        (ii) Free fatty acids: These are primarily saturated and range from 14 to 28 carbons (myristic=14, palmitic=16, stearic=18, arachidic=20, behenic=22, lignoceric=24, cerotic=26). The most common fatty acids in skin are the 22-carbon (15 wt. %) and 24 carbon (27 wt. %) species.        (iii) Triglycerides: These are minor lipid components that serve as intermediates in the transfer of fatty acids from phospholipids to glucosylceramides.        (iv) Glucosylceramides A,B,C-1,C-3,D-1,D-2, C-2: Glucosylceramide A also known as acylglucosylceramide is the major form, comprising 56 wt. % of this group of lipids. The acyl group in glucosylceramide A is often linoleic acid which is bound to the hydroxyl group of the ω-hydroxyacid (Abraham, Wertz and Downing, 1985, J. Lipid Res. 26:761–766).        (v) Ceramides 1–7: Ceramides are the major lipids in the stratum corneum. They result from deglycosylation of glucosylceramides at the end of the epidermal differentiation process. Ceramide 1 is derived from Glucosylceramide A, Ceramide 2 is derived from Glucosylceramide B, Ceramide 3 is derived from Glucosylceramide C-1, Ceramide 4 is derived from Glucosylceramide C-3, Ceramide 5 is derived from Glucosylceramide D-1, Ceramide 6-A is derived from Glucosylceramide D-2, and Ceramide 6-B is derived from Glucosylceramide C-2. Although Glucosylceramide A comprises 56 wt. % of the glucosyleramides, Ceramide 1 comprises only 8 wt. % of the extractable ceramides because most of it is converted to the (ω-hydroxyceramide which permits covalent binding to glutamates of the cornified cell envelope protein, thus, forming a protective barrier around each corneocyte. Ceramide 2 comprises 42 wt. % of this group. Ceramide 6 (phytosphingosine) and ceramide 7 (6-hydroxy-4-sphingenine) together comprise 20 wt. %.        (vi) Cholesterol: This lipid increases as keratinocytes differentiate so that it comprises 30 mol % of stratum corneum lipids (Schaefer and Redelmeier, 1996, Skin Barrier).        (vii) Cholesterol sulfate: This lipid increases cell cohesiveness by forming intercellular cholesterol sulfate calcium bridges.        (viii) Cholesterol esters: During the latter stages of epidermal differentiation, phospholipids are degraded liberating fatty acids which are utilized to produce cholesterol esters.        
Examination of lipids in serial sections through pig skin showed the following (Cox and Squier, 1986, J. Invest. Dermatol. 87:741–744): (i) increases of both glucosylceramides and ceramides towards the surface layers, but decreases of glucosylceramides concomitant with increases of ceramides at the outermost layers, (ii) decreases of phospholipids towards the surface layers of the skin (iii) progressive increases of triglycerides, cholesterol and cholesterol esters towards the surface layers of the skin, (iv) progressive increase of cholesterol sulfate and then a sudden decrease at the outermost layer (related to desquamation by a sulfatase).
Ponec and Weerheim (1990, Meth. Enzymol. 190:30–41) reviewed the literature and state that normal epidermal terminal differentiation is marked by depletion of phospholipids, with increase of sterols and certain classes of sphingolipids, with the final stratum corneum lipid products of differentiation consisting mainly of ceramides and nonpolar lipids. Overall, the flow of fatty acids during differentiation appears to be from phospholipids, to triglycerides, to ceramides, and finally hydroxy-ceramides (Swartzendruber et al., 1987, J. Invest. Dermatol. 88:709–713; Ponec et al., 1997, J. Invest. Dermatol. 109:348–355). Thus the metabolic lipid flow during differentiation appears to be towards formation of hydroxy-ceramides in the stratum corneum. Hydroxyceramides are linked to involucrin via its numerous glutamate residues (20%) during cornification, resulting in the highly effective barrier function of the skin (Swartzendruber et al., 1987, J. Invest. Dermatol. 88:709–713).
4. Agents Shown to Alter Epidermal Lipids
Retinoic acid is able to reverse the alterations of lipid synthesis that occur during differentiation, resulting in a 3–4-fold increase in phospholipids, a 3-fold decrease in sphingolipids (most notably, ceramides), a 9-fold decrease of acylceramides, a near 2-fold decrease of cholesterol and cholesterol sulfate, a 6-fold decrease of lanosterol, and a 3-fold decrease of FFA in living skin equivalents (Ponec and Weerheim, 1990, Meth. Enzymol. 190:30–41). Thus, it would appear that there are marked differences between the terminal differentiation that occurs naturally in skin, and the cellular reprogramming that occurs as a result of treatment with retinoic acid.
Vitamin C (50 ug/ml) has been found to result in increases of glucosylceramides and ceramides, most notably ceramides 6 and 7 in living skin equivalents (Ponec et al., 1997, J. Invest. Dermatol. 109:348–355). These increases were accompanied by increased barrier function. Since Vitamin E had no effect on lipid composition even though it is hydrophobic, it was concluded that the main role of Vitamin C is as a donor of hydroxyl groups to sphingoid bases and fatty acids for the formation of protein-bound hydroxyceramides (Ponec et al., 1997, J. Invest. Dermatol. 109:348–355).
5. Effect of Aging on Epidermal Lipids
All major species of epidermal lipids are decreased during the aging process. Particular attention has been paid to the reductions of the ceramide fraction since this results in a notable loss of barrier function with age (reviewed in Rogers et al., 1996, Arch. Dermatol. Res. 288:765–770). However, the percentage ratio of each of the major classes of lipids is unchanged during aging, even though total epidermal lipids are decreased by 30% in the aged (Rogers et al., 1996, Arch. Dermatol. Res. 288:765–770). The most important change of epidermal lipids that occurs with age is related to altered ratios of free fatty acids that result in reductions in ceramide 1 lineolate (Rogers et al., 1996, Arch. Dermatol. Res. 288:765–770). Reductions of ceramide 1 lineolate have been linked to dry skin, atopic dermatitis, and acne (reviewed in Rogers et al., 1996, Arch. Dermatol. Res. 288:765–770).
6. Effect of Photodamage on Epidermal Lipids
Long-term (3 week) daily treatment with either UVA (50 J/cm2) or UVB (124 mJ/cm2) has been shown to result in an approximate 2-fold increase of total epidermal lipids in human skin, with increases in the triglyceride, free fatty acid, alkane, squalene, and ceramide fractions (Wefers et al. 1991, J. Invest. Dermatol. 96:959–962). No changes were found in the sterol, cholesterol, cholesterol ester or cholesterol sulfate fractions (Wefers et al., 1991, J. Invest. Dermatol. 96:959–962). Phospholipids were not examined in this study. In contrast to these results, shortly after exposure (24–48 hr), UVA (50 J/cm2) resulted in a decrease of the ceramide fraction of living skin and an increase in the relative proportion of phospholipids (Robert et al., 1999, Int. J. Radiat. Biol. 75:317–26). Similarly, shortly after exposure (24–48 hrs), UV-B (0.15 J/cm2) resulted in a marked depletion of ceramides (Holleran et al., 1997, Photoderm. Photoimmunol. Photomed. 13:117–128). However, unlike UV-A, short-term exposure to UV-B also resulted in a marked (>2-fold) depletion of phospholipids (Holleran et al, 1997, above).
7. Methods to Treat Aged and Photodamaged Skin
Retinoic acid is well known as an agent for treatment of photoaged skin. Topical retinoic acid has been shown to restore collagen I levels that are reduced in photodamaged skin (Griffiths et al., 1993, New Engl. J. Med. 329:530–5). Restoration of collagen I levels correlate with a reduction of fine wrinkles in skin (Griffiths et al., 1993, New Engl. J. Med. 329:530–5). Although retinoids have been shown to alter lipids in cultured skin equivalents (Ponec and Weerheim, 1990, Meth. Enzymol. 190:30–41), there are no reports indicating that retinoids reverse aging or photodamage by altering lipid levels. In part, this may be because retinoids reduce ceramide levels in skin equivalents (Ponec and Weerheim, 1990, Meth. Enzymol. 190:30–41), and reduce the thickness of the stratum corneum when applied topically to human skin (Kligman and Leyden, 1993, Skin Pharmacol. 6, Suppl.1:78–82), which could exacerbate the depletion of ceramides and barrier function that occurs in the aged.
8. Pharmacological Uses of Ursolic Acid
Ursolic acid is pentacyclic triterpene compound known to have a number of pharmacological effects (reviewed in Liu, 1995, J. Ethanopharmacol. 49:57–68). Ursolic acid is closely related to steroids since both are derived from the cyclization of squalene (Suh et al., 1998, Cancer Res. 58:717–723). It is found in the waxy coating of fruit and in the leaves of many plants, such as heather and rosemary. It is insoluble in most common solvents and as a result it is not widely used. In fact, commercial extraction processes for plant leaves fail to recover measurable levels of ursolic acid.
Ursolic acid has been characterized as an inhibitor of lipoxygenase and cyclooxygenase in inflammatory cells (Najid et al., 1992, FEBS 299:213–217; Suh et al., 1998, Cancer Res. 58:717–723). As such, ursolic acid is expected to have usefulness as an anti-inflammatory agent. Ursolic acid has been shown to inhibit chronic dermal inflammation induced by phorbal esters in an animal model (Manez et al., 1997, Eur. J. Pharmacol. 334:103–105). Ursolic acid has also been shown to inhibit induction of inducible nitric oxide synthase in macrophages (Suh et al., 1998, Cancer Res. 58:717–723), which may contribute to its anti-inflammatory activity.
Ursolic acid has also been shown to induce differentiation and growth arrest of several types of cells, suggesting that it may be useful as a chemotherapeutic differentiation agent (Es-Saady et al., 1996, Cancer Lett. 106:193–197; Hsu et al., 1997, Cancer Lett. 111:7–13; Es-Saady et al., 1996, Anticancer Res. 16:481–486; Paik et al., 1998, Arch. Pharm. Res. 21:398–405). Ursolic acid has also been shown to induce apoptosis in tumor cells (Baek et al., 1997, Int. J. Cancer 73:725–728). Both ursolic acid and oleanolic acid, a closely related structural analog of ursolic acid, have been shown to inhibit tumor promotion induced in mouse skin by phorbal esters Tokuda et al., 1986, Cancer Lett. 33:279–285; Huang et al., 1994, Cancer Res. 54:701–708). Both compounds have also been shown to prevent lipid peroxidation, which may inhibit free radical damage during cancer initiation and promotion (Balanehru and Nagarajan, 1991, Biochem. Int. 24:981–990).
Ursolic acid also downregulates matrix metalloproteinases (Cha et al., 1998, Oncogene 16:771–778) and elastase (Ying et al., 1991, Biochem. J. 277:521–526) which may provide a mechanism for preventing tumor invasion (Cha et al., 1996, Cancer Res. 56:2281–84), and, inflammation related damage in skin (Ying et al., 1991, Biochem. J. 277:521–526).
Ursolic acid and a number of triterpenoid derivatives have been shown to have hypolipidemic and anti-atherosclerotic properties (reviewed in Liu, 1995, J. Ethanopharmacol. 49:57–68). Ursolic acid and oleanolic acid lowered blood cholesterol and β-lipoprotein levels 40–50% in animal models of atherosclerosis (reviewed in Liu, 1995, J. Ethanopharmacol. 49:57–68). Consistent with this prior art understanding, topical ursolic acid has been proposed for use in the treatment of psoriasis, a condition characterized by hyperproliferation and inflammation of the epidermis (U.S. Pat. No. 4,857,554). In fact, these prior results that ursolic acid and its analogs decrease lipid production and may be used in treatment of the hyperproliferation of psoriasis teach away from the present invention, and make the discovery of the opposite effects unexpected and novel. Contrary to the findings in the literature and the understanding of the prior art, we have discovered that ursolic acid increases the production of lipids, especially ceramides and phospholipids, by keratinocytes of the skin.